About the Data

Timepoints

Treat with loperamide at 5 dpf for 24 hours.

  • Sample timepoint 1 at 6 dpf (24 hour treatment)
  • Sample timepoint 2 at 7 dpf (24 hour treatment + 24 hour water)
  • Sample timepoint 3 at 11 dpf (24 hour treatment + 5 days water)

Sample collection & plating

At each timepoint:

  1. Wash all fish twice by transferring into sterile volvic in a 6-well plate
  2. Add fish with 500 µL sterile volvic water into a fastprep tube
  3. Homogenize sample at 6.5 for 45 seconds

For individual strains:
Make 0 to -3 dilutions in 96-well plates, in triplicate (8 fish per plate)
Plate 10 µL microdrops on big square plates.
8 square plates total per timepoint

Put plates at 28C for 2 days, then count colonies.

Setup

Load libraries

Import data

# import individual strain data
datacfustrial49<-
   readxl::read_xlsx("../Trial49_LoperamideCFUs/Trial49_LoperamideZebrafishWaterCFUs.xlsx", sheet="Fish") %>%
   drop_na(DF) %>%
   mutate(LoperamideTreatment=factor(LoperamideTreatment, 
                                    levels=c("None", "DMSO", "Loperamide 10 mg/L"),
                                    labels=c("Control","DMSO", "Loperamide")),
         Treatment = factor(Treatment,
                            levels=c("Bc1","Bc2","Bc3","Bc4","Bc10","Bc1/Bc2/Bc3/Bc4/Bc10")),
         Timepoint = case_when(TrialDay == "6" ~ "24 hr treatment",
                                      TrialDay == "7" ~ "Treatment +\n24 hr recovery",
                                      TrialDay == "11" ~ "Treatment +\n5 day recovery"),
         Timepoint_day = case_when(TrialDay == "6" ~ "T0",
                                      TrialDay == "7" ~ "T1",
                                      TrialDay == "11" ~ "T5"))


# import individual strain data
datacfustrial56 <-
   readxl::read_xlsx("../Trial56_LoperamideMonoCFUs/Trial56_LoperamideZebrafishMonoCFUs.xlsx", sheet="Fish") %>%
   drop_na(DF) %>%
   mutate(LoperamideTreatment=factor(LoperamideTreatment, 
                                    levels=c("None", "DMSO", "Loperamide 10 mg/L"),
                                    labels=c("Control","DMSO", "Loperamide")),
         Treatment = factor(Treatment,
                            levels=c("W6. Variovorax", "W8. Rhizobium sp.", "Mz8. Ochrobactrum"),
                            labels=c("W6","W8","Mz8")),
         Timepoint = case_when(TrialDay == "6" ~ "24 hr treatment",
                                      TrialDay == "7" ~ "Treatment +\n24 hr recovery",
                                      TrialDay == "11" ~ "Treatment +\n5 day recovery"),
         Timepoint_day = case_when(TrialDay == "6" ~ "T0",
                                      TrialDay == "7" ~ "T1",
                                      TrialDay == "11" ~ "T5"))


# import individual strain data
datacfustrial57 <-
   readxl::read_xlsx("../Trial57_LoperamideMonoMixCFUs/Trial57_LoperamideZebrafishMixMonoCFUs.xlsx", sheet="FishMono") %>%
   drop_na(DF) %>%
   mutate(LoperamideTreatment=factor(LoperamideTreatment, 
                                    levels=c("None", "DMSO", "Loperamide 10 mg/L"),
                                    labels=c("Control","DMSO", "Loperamide")),
         Treatment = factor(Treatment,
                            levels=c("Mz1. Achromobacter","F. johnsoniae"),
                            labels=c("Mz1","Fjohn")),
         Timepoint = case_when(TrialDay == "6" ~ "24 hr treatment",
                                      TrialDay == "7" ~ "Treatment +\n24 hr recovery",
                                      TrialDay == "11" ~ "Treatment +\n5 day recovery"),
         Timepoint_day = case_when(TrialDay == "6" ~ "T0",
                                      TrialDay == "7" ~ "T1",
                                      TrialDay == "11" ~ "T5"))


straininfo <- readxl::read_xlsx("../../LoperamideStrainInfo.xlsx") %>% 
   mutate(Strain=recode(Strain, "W6t"="W6"))

dataall <- full_join(datacfustrial49,datacfustrial56) %>% full_join(datacfustrial57) %>% 
   left_join(straininfo, by=c("Treatment"="Strain")) %>% 
   mutate(CodeName=factor(CodeName, levels=unique(straininfo$CodeName)))

Fish CFUs per individual strain, all timepoints together

Stats of all significant comparisons

Treatment Timepoint Timepoint_day CodeName .y. group1 group2 p p.adj p.format p.signif method
Bc1 24 hr treatment T0 S7. *Aeromonas veronii 1* CFUs_perFish DMSO Loperamide 0.04206641 1 0.042 * Wilcoxon
Bc2 24 hr treatment T0 S1. *Pseudomonas mossellii* CFUs_perFish DMSO Loperamide 0.02857143 1 0.029 * Wilcoxon
Bc2 Treatment + 24 hr recovery T1 S1. *Pseudomonas mossellii* CFUs_perFish DMSO Loperamide 0.02857143 1 0.029 * Wilcoxon
Bc1 Treatment + 5 day recovery T5 S7. *Aeromonas veronii 1* CFUs_perFish DMSO Loperamide 0.02940105 1 0.029 * Wilcoxon
Mz8 24 hr treatment T0 S9. *Ochrobactrum tritici* CFUs_perFish DMSO Loperamide 0.02857143 1 0.029 * Wilcoxon
W8 Treatment + 5 day recovery T5 S8. *Rhizobium sp.* CFUs_perFish DMSO Loperamide 0.02857143 1 0.029 * Wilcoxon
Fjohn 24 hr treatment T0 S10. *Flavobacterium johnsoniae* CFUs_perFish DMSO Loperamide 0.02842954 1 0.028 * Wilcoxon
Fjohn Treatment + 24 hr recovery T1 S10. *Flavobacterium johnsoniae* CFUs_perFish DMSO Loperamide 0.02940105 1 0.029 * Wilcoxon

First timepoint, total CFUs per Fish

Faceted by Strain, with means shown.

Stats are relative to DMSO

Timeline for each strain with mean

Timeline for each strain with boxplots

Timeline for each strain with boxplots - formatted

Timeline for control conditions

Compare with Water CFUs